{"gene":"IGSF8","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2001,"finding":"EWI-2 (IGSF8) was co-purified with tetraspanin CD81 under stringent detergent conditions and identified by mass spectrometry. It associates specifically and directly with CD9 and CD81 (but not other tetraspanins or integrins) in a highly stoichiometric manner (~70% of CD9 and CD81 associated with EWI-2), as confirmed by covalent cross-linking experiments.","method":"Co-purification under Brij 96 detergent, mass spectrometry, immunodepletion, chemical cross-linking","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-purification with mass spectrometric identification and cross-linking confirmation, replicated across multiple experiments and cell lines","pmids":["11504738"],"is_preprint":false},{"year":2001,"finding":"The interaction of CD81 with PGRL (EWI-2/IGSF8) requires the membrane-distal Ig3-Ig4 domains of PGRL, as determined by domain deletion constructs co-expressed with CD81.","method":"Co-expression of domain deletion constructs with CD81, immunoprecipitation","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping using epitope-tagged deletion constructs, single lab","pmids":["11673522"],"is_preprint":false},{"year":2003,"finding":"Two separate regions of CD9 (40 and 47 amino acids) confer the ability to interact with EWI-2, as determined using chimeric CD9/CD82 molecules. EWI-2 was also shown to associate with CD81 on freshly isolated hepatocytes and to co-localize with CD81 in the liver.","method":"Chimeric CD9/CD82 protein expression, co-immunoprecipitation, immunofluorescence co-localization","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping with chimeric proteins, single lab, orthogonal localization data","pmids":["12708969"],"is_preprint":false},{"year":2003,"finding":"EWI-2/PGRL (IGSF8) physically associates with the metastasis suppressor KAI1/CD82 in a highly stoichiometric, direct complex independent of cholesterol-enriched lipid rafts and distinct from CD81/CD9 complexes. Overexpression of EWI-2/PGRL in Du145 prostate cancer cells inhibits cell migration on fibronectin- and laminin-coated surfaces, and synergizes with KAI1/CD82 in migration suppression.","method":"Co-immunoprecipitation, chemical cross-linking, cell migration assays, overexpression","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP with cross-linking and functional migration assay, single lab","pmids":["12750295"],"is_preprint":false},{"year":2003,"finding":"EWI-2 overexpression in A431 cells impairs cell reaggregation and motility on laminin-5 (an alpha3beta1 integrin ligand) but not on collagen I. Tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin. CD81 controls EWI-2 maturation and cell surface localization. A chimeric EWI-2 mutant failed to suppress migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation.","method":"Overexpression, immunodepletion, reexpression experiments, cell migration assays, mutagenesis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (immunodepletion, overexpression, mutant rescue, functional assays), mechanistic pathway placement established","pmids":["14662754"],"is_preprint":false},{"year":2003,"finding":"EWI-2 is the most prominent alpha4beta1 integrin partner on MOLT-4 T leukemia cells. Wild-type EWI-2 overexpression markedly impairs cell spreading and ruffling on VCAM-1, while a cytoplasmic tail mutant EWI-2 neither impairs spreading nor associates with alpha4beta1 or CD81. EWI-2 overexpression reorganizes cell-surface CD81, increases CD81-CD81, CD81-alpha4beta1, and alpha4beta1-alpha4beta1 associations, and increases the apparent size of CD81-alpha4beta1 complexes.","method":"Anti-integrin antibody bead isolation, mass spectrometry, overexpression, cytoplasmic tail mutagenesis, shear flow adhesion assays, size exclusion chromatography","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — mass spectrometric identification, multiple functional assays, mutagenesis confirming cytoplasmic tail requirement, single lab with orthogonal methods","pmids":["15070678"],"is_preprint":false},{"year":2006,"finding":"EWI-2 (IGSF8) directly associates with ezrin-radixin-moesin (ERM) proteins through a basic charged amino acid stretch in its cytoplasmic domain. EWI-2 co-localizes with ERM proteins at microspikes, microvilli, and the cellular uropod in polarized leukocytes. Silencing EWI-2 augments cell migration, cellular polarity, and increases ERM phosphorylation. A dominant-negative moesin N-terminal domain delocalizes EWI-2 from the uropod. EWI-2 thereby links tetraspanin microdomains to the actin cytoskeleton.","method":"Co-immunoprecipitation, GST pulldown, confocal microscopy, siRNA knockdown, dominant-negative overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding confirmed by GST pulldown, in vivo co-IP, dominant-negative functional validation, and siRNA phenotype with multiple orthogonal methods","pmids":["16690612"],"is_preprint":false},{"year":2007,"finding":"EWI-2/CD316 (IGSF8) functions as a receptor for heat shock protein HSPA8 (hsp70 family member) on human dendritic cells. Soluble EWI-2 bound to cells expressing HSPA8 and to immobilized HSPA8 protein. HSPA8 ligation of EWI-2 enhanced CCL21/SLC-dependent migration of mature dendritic cells but attenuated their antigen-specific stimulatory capacity.","method":"Expression cloning, binding assays (soluble protein to cells and immobilized protein), functional migration assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — expression cloning identification of ligand plus direct binding assays and functional readouts, single lab","pmids":["17785435"],"is_preprint":false},{"year":2009,"finding":"EWI-2 (IGSF8) overexpression in glioblastoma cell lines inhibits colony formation in soft agar and reduces cell motility and invasion. At the biochemical level, EWI-2 causes CD9 and CD81 to become more associated with each other and causes CD81 and other tetraspanins to become less associated with MMP-2 and MT1-MMP.","method":"Overexpression in T98G and U87-MG cells, soft agar assay, motility/invasion assays, co-immunoprecipitation, in vivo xenograft","journal":"Neoplasia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays plus co-IP evidence for molecular reorganization, single lab","pmids":["19107234"],"is_preprint":false},{"year":2009,"finding":"EWI-2 (IGSF8) forms a direct, stable interaction with CD9 on mouse eggs (stable in 1% Triton X-100). Anti-IgSF8 antibody has moderate inhibitory effects on sperm-egg binding, whereas anti-CD9 antibody significantly inhibits sperm-egg fusion, indicating that IgSF8 and CD9 have discrete, non-identical functions in gamete interaction.","method":"Detergent co-immunoprecipitation, antibody perturbation in IVF assays","journal":"Reproduction, fertility, and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct co-IP under stringent detergent conditions and antibody functional assays with distinct phenotypic readouts","pmids":["19210920"],"is_preprint":false},{"year":2009,"finding":"On CD9-null oocytes, surface EWI-2 expression is reduced to <10% of wild-type levels, indicating that CD9 is required for normal surface localization of EWI-2. Depalmitoylated CD9 mutant does not affect EWI-2 surface levels, showing that CD9 palmitoylation is not required for maintaining EWI-2 at the surface.","method":"Flow cytometry on CD9 knockout oocytes, depalmitoylation mutant analysis","journal":"Molecular reproduction and development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with quantitative surface protein measurement, single lab","pmids":["19107828"],"is_preprint":false},{"year":2011,"finding":"A glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint is essential for interaction with CD81. Palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for interaction with both CD81 and CD9. EWI-2wint (a proteolytic cleavage product of EWI-2) must interact with CD81 to exert its inhibitory effect on HCV infection.","method":"Mutagenesis of transmembrane glycine zipper and cytoplasmic cysteine residues, co-immunoprecipitation, HCV infection functional assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis of specific motifs (glycine zipper, palmitoylation cysteines) with co-IP and functional HCV infection assays; multiple orthogonal mechanistic validations in single study","pmids":["21343309"],"is_preprint":false},{"year":2011,"finding":"The EWI-2 cytoplasmic tail specifically interacts with negatively charged phosphatidylinositol phosphates (PIPs), particularly PtdIns5P, PtdIns4P, and PtdIns3P, but not other membrane lipids. Mutation of either of two basic residue clusters abolishes PIP binding. EWI-2 is constitutively palmitoylated at cytoplasmic cysteine residues. PIP binding regulates EWI-2 protein stability, while palmitoylation is required for tetraspanin-EWI-2 association and EWI-2-dependent inhibition of cell migration and lamellipodia formation.","method":"Lipid binding assays, site-directed mutagenesis of basic residue clusters and cysteines, palmitoylation assays, cell migration assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro lipid binding assays with mutagenesis, palmitoylation assays, and functional cellular readouts establishing differential roles of two modifications","pmids":["21609323"],"is_preprint":false},{"year":2012,"finding":"EWI-2 co-localizes with CD3 and CD81 at the central supramolecular activation cluster (cSMAC) of the T cell immune synapse. Mass spectrometry of pull-downs with the C-terminal intracellular domain of EWI-2 identified α-actinin as a specific binding partner; this association is regulated by PIP2. Silencing EWI-2 or overexpression of a cytoplasmic-truncated EWI-2 mutant increases IL-2 secretion upon antigen stimulation. Silencing EWI-2 or α-actinin-4 increases HIV cell infectivity at virological synapses.","method":"Confocal microscopy, mass spectrometry pull-down, siRNA knockdown, overexpression of truncation mutant, IL-2 secretion assay, HIV infectivity assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — mass spectrometric identification of binding partner, co-localization, mutagenesis, and multiple functional readouts (IL-2, HIV infection), single lab with orthogonal methods","pmids":["22689882"],"is_preprint":false},{"year":2012,"finding":"IGSF8 (EWI-2) interacts with tetraspanin CD9 in the olfactory bulb. Both IgSF8 and CD9 localize to puncta within axons and growth cones of olfactory sensory neurons, consistent with tetraspanin-enriched microdomain localization. IgSF8 expression in synaptic neuropil is transitory during glomerular formation and re-appears upon OSN regeneration, consistent with a role in synapse formation.","method":"Co-immunoprecipitation from olfactory bulb tissue, immunofluorescence, in vivo lesion/regeneration experiments","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP from tissue, in vivo regeneration experiment, single lab","pmids":["22687584"],"is_preprint":false},{"year":2012,"finding":"Igsf8-deficient female mice show no fertilization defect in vitro or in vivo. Igsf8-deficient eggs retain normal CD9 levels and localization with normal microvilli formation, indicating IGSF8 is dispensable for fertility and not required for CD9 surface localization (contrary to a previous suggestion).","method":"Homologous recombination knockout, in vitro and in vivo fertility testing, immunofluorescence","journal":"Fertility and sterility","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with in vivo and in vitro functional tests; negative result rigorously established","pmids":["22609062"],"is_preprint":false},{"year":2015,"finding":"EWI-2 (IGSF8) negatively regulates TGF-β signaling in melanoma. When EWI-2 is present, it sequesters CD9 and CD81 away from TGF-β receptors. When EWI-2 is knocked down, CD9 and CD81 become available to support TβR2-TβR1 association, elevating TGF-β signaling and downstream cytostasis, EMT-like changes, CD271-dependent invasion, and lung metastasis. All EWI-2 depletion phenotypes are reversed by blocking or depleting CD9 or CD81.","method":"RNAi knockdown, overexpression, co-immunoprecipitation, in vitro and in vivo functional assays, epistasis by CD9/CD81 blocking","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis established by rescue experiments (CD9/CD81 depletion reverses EWI-2 KD phenotype), multiple orthogonal functional readouts in vitro and in vivo","pmids":["25656846"],"is_preprint":false},{"year":2019,"finding":"EWI-2 accumulates at the presynaptic terminal (producer cell side) of the HIV virological synapse and contributes to inhibition of Env-mediated cell-cell fusion. EWI-2 is downregulated upon HIV-1 infection, likely by Vpu. EWI-2 and CD81 surface levels are restored on syncytia where they continue to act as fusion inhibitors.","method":"Quantitative fluorescence microscopy, shRNA knockdown, cell-cell fusion assays","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — shRNA knockdown with cell-cell fusion functional assay and quantitative microscopy, single lab","pmids":["31757023"],"is_preprint":false},{"year":2020,"finding":"IgSF8 is a neuronal receptor enriched in the hippocampal mossy fiber (MF) pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Cell-surface interactome screening identified IgSF8 interaction partners at the MF synapse.","method":"MF synaptosome proteomic profiling, cell-surface interactome screening, conditional presynaptic knockout, electrophysiology, electron microscopy","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with multiple orthogonal readouts (structural, electrophysiological), interactome screening, single study with rigorous controls","pmids":["33057002"],"is_preprint":false},{"year":2021,"finding":"EWI-2 (IGSF8) localizes not only on the plasma membrane but also on the nuclear envelope, where it regulates nuclear translocation of EGFR-MAPK-ERK signaling molecules and sorting of miR-3934-5p between cells and exosomes, thereby regulating prostate cancer cell metastasis.","method":"Subcellular fractionation, immunofluorescence, exosome isolation, miRNA quantification, signaling pathway analysis","journal":"Molecular oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, nuclear localization claim and miRNA sorting based on fractionation without functional mutagenesis; no mechanistic dissection of how nuclear EWI-2 regulates these processes","pmids":["33605506"],"is_preprint":false},{"year":2022,"finding":"EWI2 (IGSF8) prevents EGFR from clustering and endocytosis, thereby restraining EGFR activation and ERK MAP kinase signaling. Upon EWI2 silencing or ablation, EGFR clustering, endocytosis, and activation increase, leading to enhanced cancer cell proliferation and migration. Inhibition of EGFR or ERK kinase abrogates the phenotypes caused by EWI2 removal.","method":"siRNA knockdown, CRISPR ablation, super-resolution microscopy (EGFR clustering), endocytosis assays, kinase inhibitor epistasis, in vivo xenograft","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR knockout plus pharmacological epistasis, direct measurement of EGFR clustering, single lab","pmids":["35773608"],"is_preprint":false},{"year":2022,"finding":"EWI2 (IGSF8) promotes endolysosomal trafficking and lysosomal degradation of growth factor receptors and integrins by promoting nuclear retention of the master transcription factor TFEB, thereby driving lysosomogenesis and reducing surface levels of these receptors in lung cancer cells.","method":"Forced overexpression, lysosomal trafficking assays, TFEB nuclear localization experiments, receptor degradation assays, in vivo tumor models","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic linkage via TFEB nuclear retention with functional lysosomal degradation readouts, single lab","pmids":["35339615"],"is_preprint":false},{"year":2024,"finding":"IGSF8 expressed on tumors suppresses NK cell function by directly interacting with human KIR3DL2 and mouse Klra9 inhibitory receptors on NK cells. An antibody blocking IGSF8-NK receptor interaction enhances NK cell killing of malignant cells in vitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and T cell signaling in vivo. Anti-IGSF8 alone or combined with anti-PD1 inhibits tumor growth in syngeneic models.","method":"CRISPR screens, co-immunoprecipitation/binding assays, antibody blockade, NK cell killing assays, in vivo syngeneic tumor models","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen discovery followed by direct binding confirmation, antibody functional blockade, and in vivo validation in multiple tumor models","pmids":["38657602"],"is_preprint":false}],"current_model":"IGSF8 (EWI-2) is a cell-surface immunoglobulin superfamily transmembrane protein that forms highly stoichiometric, direct complexes with tetraspanins CD9 and CD81 (requiring its Ig3-Ig4 domains and cytoplasmic palmitoylation), links tetraspanin-enriched microdomains to the actin cytoskeleton via direct binding to ERM proteins and α-actinin through its basic cytoplasmic tail, regulates integrin (α3β1, α4β1) and EGFR clustering and signaling, suppresses TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors, promotes lysosomal degradation of surface receptors via TFEB nuclear retention, inhibits HIV cell-cell fusion at virological synapses, functions as a presynaptic organizer of hippocampal CA3 mossy fiber microcircuit connectivity, and serves as an innate immune checkpoint by engaging inhibitory KIR3DL2/Klra9 receptors on NK cells to suppress anti-tumor cytotoxicity."},"narrative":{"mechanistic_narrative":"IGSF8 (EWI-2) is a cell-surface immunoglobulin-superfamily transmembrane protein that organizes tetraspanin-enriched microdomains and couples them to the actin cytoskeleton and downstream receptor signaling [PMID:11504738, PMID:16690612]. It forms highly stoichiometric, direct, cross-linkable complexes specifically with the tetraspanins CD9 and CD81 (~70% of CD9/CD81 engaged), an interaction that requires its membrane-distal Ig3-Ig4 domains, a transmembrane glycine-zipper motif, and palmitoylation of juxtamembrane cytoplasmic cysteines [PMID:11504738, PMID:11673522, PMID:21343309]. Through these tetraspanins IGSF8 is physically linked to integrins (α3β1, α4β1), and its overexpression reorganizes surface CD81-integrin complexes and suppresses cell spreading, migration, and invasion in a manner dependent on its cytoplasmic tail [PMID:14662754, PMID:15070678]. The basic cytoplasmic tail binds ERM proteins and α-actinin and engages phosphatidylinositol phosphates, tethering tetraspanin microdomains to the cytoskeleton at uropods and immune synapses; PIP binding stabilizes the protein while palmitoylation enables tetraspanin association and migration suppression [PMID:16690612, PMID:21609323, PMID:22689882]. IGSF8 restrains receptor signaling on multiple axes: it sequesters CD9/CD81 away from TGF-β receptors to limit TGF-β-driven EMT and metastasis [PMID:25656846], prevents EGFR clustering and endocytosis to dampen ERK signaling [PMID:35773608], and promotes lysosomal degradation of growth-factor receptors and integrins via TFEB nuclear retention [PMID:35339615]. It localizes to the central supramolecular activation cluster of the T-cell synapse and to HIV virological synapses, where it inhibits Env-mediated cell-cell fusion [PMID:22689882, PMID:31757023]. In neurons, IGSF8 acts as a presynaptic organizer of hippocampal mossy-fiber connectivity, controlling bouton filopodia and excitation/inhibition balance [PMID:33057002]. As a tumor-expressed ligand it engages the inhibitory NK-cell receptors KIR3DL2 (human) and Klra9 (mouse) to suppress anti-tumor cytotoxicity, and antibody blockade of this interaction enhances NK killing and tumor control [PMID:38657602].","teleology":[{"year":2001,"claim":"Established the founding molecular fact that IGSF8 is a dedicated, near-stoichiometric direct partner of specific tetraspanins, defining it as a core component of tetraspanin microdomains rather than a promiscuous associate.","evidence":"Co-purification under stringent Brij 96 detergent, mass spectrometry, and chemical cross-linking; domain-deletion mapping with CD81","pmids":["11504738","11673522"],"confidence":"High","gaps":["No structure of the IGSF8-tetraspanin complex","Stoichiometry of higher-order assemblies undefined"]},{"year":2003,"claim":"Mapped the binding determinants on the tetraspanin side and showed IGSF8 also partners the metastasis suppressor CD82, linking tetraspanin association to suppression of tumor cell migration.","evidence":"Chimeric CD9/CD82 constructs, reciprocal co-IP with cross-linking, hepatocyte co-localization, and migration assays in prostate cancer cells","pmids":["12708969","12750295"],"confidence":"Medium","gaps":["Mechanism of migration suppression by IGSF8-CD82 not resolved","Single-lab functional assays"]},{"year":2003,"claim":"Demonstrated that IGSF8 reorganizes integrin-tetraspanin complexes and that this activity, and surface maturation, depend on CD81 and on the IGSF8 cytoplasmic tail.","evidence":"Overexpression, immunodepletion/reexpression, chimeric and cytoplasmic-tail mutants, migration and shear-flow adhesion assays for α3β1 and α4β1","pmids":["14662754","15070678"],"confidence":"High","gaps":["Direct vs tetraspanin-bridged contact with integrins not separated","Signaling consequences downstream of integrin clustering undefined"]},{"year":2006,"claim":"Identified the cytoskeletal coupling mechanism, showing the basic cytoplasmic tail directly binds ERM proteins to tether tetraspanin microdomains to actin and restrain migration and polarity.","evidence":"GST pulldown, co-IP, confocal microscopy, siRNA knockdown, dominant-negative moesin in polarized leukocytes","pmids":["16690612"],"confidence":"High","gaps":["How ERM binding is regulated by activation state unknown","Relationship between ERM binding and integrin reorganization unresolved"]},{"year":2007,"claim":"Proposed IGSF8 as a surface receptor for HSPA8 on dendritic cells with dual effects on migration and antigen presentation, broadening its role beyond tetraspanin scaffolding.","evidence":"Expression cloning, soluble-protein binding assays, dendritic cell migration and stimulation assays","pmids":["17785435"],"confidence":"Medium","gaps":["Binding site and physiological relevance of HSPA8 engagement unconfirmed","Single-lab finding not extended"]},{"year":2009,"claim":"Defined IGSF8-CD9 complexes in gametes and tested their reproductive role, with conflicting early conclusions about whether IGSF8 controls CD9 surface localization.","evidence":"Stringent detergent co-IP, antibody perturbation in IVF, flow cytometry on CD9-null oocytes","pmids":["19210920","19107828"],"confidence":"Medium","gaps":["Apparent dependence of IGSF8 on CD9 later contradicted by knockout data","Modest antibody effects on sperm-egg binding hard to interpret"]},{"year":2009,"claim":"Showed IGSF8 overexpression suppresses glioblastoma colony formation and invasion by tightening CD9/CD81 association and reducing tetraspanin-MMP coupling, generalizing its tumor-suppressive microdomain remodeling.","evidence":"Overexpression in glioma lines, soft agar, motility/invasion assays, co-IP, xenografts","pmids":["19107234"],"confidence":"Medium","gaps":["Direct effect on MMP activity not measured","Single-lab functional data"]},{"year":2011,"claim":"Resolved the molecular grammar of tetraspanin binding and connected a proteolytic IGSF8 product to viral entry inhibition, defining the transmembrane and palmitoylation requirements.","evidence":"Mutagenesis of the transmembrane glycine zipper and juxtamembrane palmitoylation cysteines, co-IP, HCV infection assays; PIP-binding lipid assays and palmitoylation assays","pmids":["21343309","21609323"],"confidence":"High","gaps":["How EWI-2wint cleavage is regulated unknown","Distinct contributions of PIP binding vs palmitoylation to in vivo function not fully separated"]},{"year":2012,"claim":"Placed IGSF8 at the immune and virological synapse via α-actinin/PIP2-regulated cytoskeletal coupling, where it dampens T-cell IL-2 output and restricts HIV infectivity.","evidence":"Confocal microscopy, mass-spectrometry pulldown identifying α-actinin, siRNA, truncation mutants, IL-2 and HIV infectivity assays; plus olfactory-bulb co-IP and regeneration data","pmids":["22689882","22687584"],"confidence":"High","gaps":["Direct vs indirect α-actinin contact in vivo unconfirmed","Mechanism connecting synapse architecture to cytokine output unresolved"]},{"year":2012,"claim":"Genetic knockout established that IGSF8 is dispensable for fertility and not required for CD9 surface localization, correcting an earlier interpretation.","evidence":"Homologous-recombination knockout with in vitro and in vivo fertility testing and immunofluorescence","pmids":["22609062"],"confidence":"High","gaps":["Does not address redundancy with other EWI proteins","Reproductive role, if any, remains negative"]},{"year":2015,"claim":"Established a defined signaling mechanism: IGSF8 suppresses TGF-β signaling and metastasis by sequestering CD9/CD81 from TGF-β receptors, proven by CD9/CD81-dependent rescue.","evidence":"RNAi, overexpression, co-IP, epistasis via CD9/CD81 blockade, in vitro and in vivo melanoma metastasis assays","pmids":["25656846"],"confidence":"High","gaps":["Quantitative basis of tetraspanin partitioning between receptor pools undefined","Generality across other TGF-β receptor contexts untested"]},{"year":2019,"claim":"Extended the fusion-inhibitory role to the HIV producer cell, showing IGSF8 is downregulated during infection (likely by Vpu) and inhibits Env-mediated cell-cell fusion.","evidence":"Quantitative fluorescence microscopy, shRNA, cell-cell fusion assays","pmids":["31757023"],"confidence":"Medium","gaps":["Direct mechanism of Vpu-mediated downregulation not demonstrated","Single-lab functional data"]},{"year":2020,"claim":"Defined a developmental neuronal function: presynaptic IGSF8 organizes mossy-fiber microcircuit architecture and excitation/inhibition balance.","evidence":"MF synaptosome proteomics, cell-surface interactome screening, conditional presynaptic knockout, electrophysiology, electron microscopy","pmids":["33057002"],"confidence":"High","gaps":["Trans-synaptic ligand mediating the organizing activity not pinpointed","Relationship to tetraspanin partners in neurons unresolved"]},{"year":2022,"claim":"Unified IGSF8's receptor-restraining activities, showing it limits EGFR clustering/endocytosis and drives TFEB-dependent lysosomal degradation of growth-factor receptors and integrins to suppress tumor cell proliferation.","evidence":"siRNA/CRISPR ablation, super-resolution imaging of EGFR clustering, endocytosis assays, kinase-inhibitor epistasis, TFEB nuclear-localization and receptor-degradation assays, xenografts","pmids":["35773608","35339615"],"confidence":"Medium","gaps":["How IGSF8 signals to TFEB nuclear retention is unknown","Direct mechanism preventing EGFR clustering not structurally defined"]},{"year":2024,"claim":"Identified IGSF8 as a tumor-expressed innate immune checkpoint engaging inhibitory NK receptors KIR3DL2/Klra9, establishing a therapeutically actionable ligand-receptor axis.","evidence":"CRISPR screens, binding/co-IP, antibody blockade, NK killing assays, syngeneic tumor models with anti-PD1 combination","pmids":["38657602"],"confidence":"High","gaps":["Structural basis of IGSF8-KIR3DL2 recognition not defined","Relationship between checkpoint function and tetraspanin scaffolding unexplored"]},{"year":null,"claim":"How IGSF8's diverse roles—tetraspanin scaffolding, cytoskeletal coupling, receptor signaling restraint, synaptic organization, and NK checkpoint ligand activity—are integrated by a single protein, and how these are regulated in different cell types, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of full-length IGSF8 in complex with any partner","Whether the extracellular NK-receptor engagement and intracellular tetraspanin/cytoskeleton functions act independently is unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[16,20,21]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[6,13]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[12]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[22]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[7,22]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4,13]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[6,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[16,20]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[13,22]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[16,22]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[20,21]}],"complexes":["tetraspanin-enriched microdomain (CD9/CD81/EWI-2)"],"partners":["CD81","CD9","CD82","ERM PROTEINS","ALPHA-ACTININ","KIR3DL2","HSPA8","ITGA3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q969P0","full_name":"Immunoglobulin superfamily member 8","aliases":["CD81 partner 3","Glu-Trp-Ile EWI motif-containing protein 2","EWI-2","Keratinocytes-associated transmembrane protein 4","KCT-4","LIR-D1","Prostaglandin regulatory-like protein","PGRL"],"length_aa":613,"mass_kda":65.0,"function":"Member of the immunoglobulin superfamily (IgSF) that links tetraspanin-enriched microdomains to the actin cytoskeleton and plays several important roles in innate and adaptive immunity (PubMed:11504738, PubMed:14662754). Acts as an inducible receptor of HSPA8 on dendritic cells to enhance the CCL21/SLC-dependent migration of activated mature dendritic cells while attenuating their antigen-specific stimulatory capacities (PubMed:17785435). In complex with alpha-actinins ACTN1 and ACTN4, regulates actin dynamics in the immune synapse and subsequent T-cell activation (PubMed:22689882). Inhibits the entry of several viruses such as hepatitis C Virus (HCV) or HIV-1. Mechanistically, promotes a change in CD81 organization at the plasma membrane by significantly restricting its diffusion which in turn influences CD81 interaction with Claudin-1/CLDN1, preventing CLDN1 from acting as a co-receptor required for HCV entry (PubMed:23351194). Accumulates at the presynaptic terminal, the producer cell side of the virological synapse, to prevent HIV-1 Env-mediated cell-cell fusion (PubMed:31757023). Highly expressed on malignant cells with antigen presentation defects, interacts with NK receptor KIR3DL2 to suppress NK-cell cytotoxicity (PubMed:38657602). May participate in the regulation of neurite outgrowth and maintenance of the neural network in the adult brain","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q969P0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IGSF8","classification":"Not Classified","n_dependent_lines":14,"n_total_lines":1208,"dependency_fraction":0.011589403973509934},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/IGSF8","total_profiled":1310},"omim":[{"mim_id":"606644","title":"IMMUNOGLOBULIN SUPERFAMILY, MEMBER 8; IGSF8","url":"https://www.omim.org/entry/606644"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":153.9}],"url":"https://www.proteinatlas.org/search/IGSF8"},"hgnc":{"alias_symbol":["CD81P3","EWI2","PGRL","CD316"],"prev_symbol":[]},"alphafold":{"accession":"Q969P0","domains":[{"cath_id":"2.60.40.10","chopping":"28-148","consensus_level":"high","plddt":95.1681,"start":28,"end":148},{"cath_id":"2.60.40.10","chopping":"152-160_171-298","consensus_level":"high","plddt":93.3626,"start":152,"end":298},{"cath_id":"2.60.40.10","chopping":"305-433","consensus_level":"high","plddt":90.9349,"start":305,"end":433},{"cath_id":"2.60.40.10","chopping":"439-483_494-572","consensus_level":"high","plddt":89.969,"start":439,"end":572}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969P0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q969P0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q969P0-F1-predicted_aligned_error_v6.png","plddt_mean":88.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IGSF8","jax_strain_url":"https://www.jax.org/strain/search?query=IGSF8"},"sequence":{"accession":"Q969P0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q969P0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q969P0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969P0"}},"corpus_meta":[{"pmid":"11504738","id":"PMC_11504738","title":"EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11504738","citation_count":190,"is_preprint":false},{"pmid":"16690612","id":"PMC_16690612","title":"EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16690612","citation_count":165,"is_preprint":false},{"pmid":"12708969","id":"PMC_12708969","title":"EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells.","date":"2003","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/12708969","citation_count":117,"is_preprint":false},{"pmid":"12750295","id":"PMC_12750295","title":"EWI2/PGRL associates with the metastasis suppressor KAI1/CD82 and inhibits the migration of prostate cancer cells.","date":"2003","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/12750295","citation_count":88,"is_preprint":false},{"pmid":"11673522","id":"PMC_11673522","title":"PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins.","date":"2001","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/11673522","citation_count":74,"is_preprint":false},{"pmid":"14662754","id":"PMC_14662754","title":"EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5.","date":"2003","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/14662754","citation_count":72,"is_preprint":false},{"pmid":"15070678","id":"PMC_15070678","title":"EWI-2 modulates lymphocyte integrin alpha4beta1 functions.","date":"2003","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15070678","citation_count":51,"is_preprint":false},{"pmid":"21343309","id":"PMC_21343309","title":"Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21343309","citation_count":50,"is_preprint":false},{"pmid":"25656846","id":"PMC_25656846","title":"EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis.","date":"2015","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/25656846","citation_count":48,"is_preprint":false},{"pmid":"33057002","id":"PMC_33057002","title":"Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33057002","citation_count":45,"is_preprint":false},{"pmid":"22689882","id":"PMC_22689882","title":"EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection.","date":"2012","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/22689882","citation_count":44,"is_preprint":false},{"pmid":"38657602","id":"PMC_38657602","title":"IGSF8 is an innate immune checkpoint and cancer immunotherapy target.","date":"2024","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/38657602","citation_count":43,"is_preprint":false},{"pmid":"19107234","id":"PMC_19107234","title":"Glioblastoma inhibition by cell surface immunoglobulin protein EWI-2, in vitro and in vivo.","date":"2009","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/19107234","citation_count":42,"is_preprint":false},{"pmid":"19210920","id":"PMC_19210920","title":"Immunoglobulin superfamily member IgSF8 (EWI-2) and CD9 in fertilisation: evidence of distinct functions for CD9 and a CD9-associated protein in mammalian sperm-egg interaction.","date":"2009","source":"Reproduction, fertility, and development","url":"https://pubmed.ncbi.nlm.nih.gov/19210920","citation_count":32,"is_preprint":false},{"pmid":"33605506","id":"PMC_33605506","title":"EWI-2 controls nucleocytoplasmic shuttling of EGFR signaling molecules and miRNA sorting in exosomes to inhibit prostate cancer cell metastasis.","date":"2021","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33605506","citation_count":21,"is_preprint":false},{"pmid":"17785435","id":"PMC_17785435","title":"EWI-2/CD316 is an inducible receptor of HSPA8 on human dendritic cells.","date":"2007","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17785435","citation_count":20,"is_preprint":false},{"pmid":"22609062","id":"PMC_22609062","title":"Tetraspanin-interacting protein IGSF8 is dispensable for mouse fertility.","date":"2012","source":"Fertility and sterility","url":"https://pubmed.ncbi.nlm.nih.gov/22609062","citation_count":17,"is_preprint":false},{"pmid":"21609323","id":"PMC_21609323","title":"Differential functions of phospholipid binding and palmitoylation of tumour suppressor EWI2/PGRL.","date":"2011","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/21609323","citation_count":16,"is_preprint":false},{"pmid":"26989766","id":"PMC_26989766","title":"Novel impact of EWI-2, CD9, and CD81 on TGF-β signaling in melanoma.","date":"2015","source":"Molecular & cellular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/26989766","citation_count":16,"is_preprint":false},{"pmid":"22687584","id":"PMC_22687584","title":"IgSF8: a developmentally and functionally regulated cell adhesion molecule in olfactory sensory neuron axons and synapses.","date":"2012","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/22687584","citation_count":15,"is_preprint":false},{"pmid":"31757023","id":"PMC_31757023","title":"EWI-2 Inhibits Cell-Cell Fusion at the HIV-1 Virological Presynapse.","date":"2019","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/31757023","citation_count":13,"is_preprint":false},{"pmid":"19107828","id":"PMC_19107828","title":"Loss of surface EWI-2 on CD9 null oocytes.","date":"2009","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/19107828","citation_count":12,"is_preprint":false},{"pmid":"36788350","id":"PMC_36788350","title":"EWI2 and its relatives in Tetraspanin-enriched membrane domains regulate malignancy.","date":"2023","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/36788350","citation_count":11,"is_preprint":false},{"pmid":"12595239","id":"PMC_12595239","title":"Genomic organization and embryonic expression of Igsf8, an immunoglobulin superfamily member implicated in development of the nervous system and organ epithelia.","date":"2003","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/12595239","citation_count":10,"is_preprint":false},{"pmid":"35773608","id":"PMC_35773608","title":"EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.","date":"2022","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/35773608","citation_count":8,"is_preprint":false},{"pmid":"35339615","id":"PMC_35339615","title":"EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.","date":"2022","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/35339615","citation_count":6,"is_preprint":false},{"pmid":"38453613","id":"PMC_38453613","title":"IGSF8 is a potential target for the treatment of gliomas.","date":"2024","source":"Asian journal of surgery","url":"https://pubmed.ncbi.nlm.nih.gov/38453613","citation_count":5,"is_preprint":false},{"pmid":"28544021","id":"PMC_28544021","title":"Differential expression of EWI-2 in endometriosis, its functional role and underlying molecular mechanisms.","date":"2017","source":"The journal of obstetrics and gynaecology research","url":"https://pubmed.ncbi.nlm.nih.gov/28544021","citation_count":4,"is_preprint":false},{"pmid":"39755227","id":"PMC_39755227","title":"IGSF8 impairs migration and invasion of trophoblast cells and angiogenesis in preeclampsia.","date":"2025","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/39755227","citation_count":2,"is_preprint":false},{"pmid":"1840028","id":"PMC_1840028","title":"Thymic origin of some natural killer cells: clonal proliferation of human CD3-16+ cells from CD3-4-8- thymocyte precursors requires the presence of H9 leukemic cells.","date":"1991","source":"International journal of clinical & laboratory research","url":"https://pubmed.ncbi.nlm.nih.gov/1840028","citation_count":1,"is_preprint":false},{"pmid":"40936932","id":"PMC_40936932","title":"Functional characterization and clinical significance of IGSF8 in pan-cancer: an integrated bioinformatic and experimental study.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40936932","citation_count":0,"is_preprint":false},{"pmid":"41346582","id":"PMC_41346582","title":"R3HDM4 influences kidney renal clear cell carcinoma progression, immune modulation, and potential links to the IGSF8 immune checkpoint.","date":"2025","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41346582","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17682,"output_tokens":6198,"usd":0.073008,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14877,"output_tokens":5209,"usd":0.102305,"stage2_stop_reason":"end_turn"},"total_usd":0.175313,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"EWI-2 (IGSF8) was co-purified with tetraspanin CD81 under stringent detergent conditions and identified by mass spectrometry. It associates specifically and directly with CD9 and CD81 (but not other tetraspanins or integrins) in a highly stoichiometric manner (~70% of CD9 and CD81 associated with EWI-2), as confirmed by covalent cross-linking experiments.\",\n      \"method\": \"Co-purification under Brij 96 detergent, mass spectrometry, immunodepletion, chemical cross-linking\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-purification with mass spectrometric identification and cross-linking confirmation, replicated across multiple experiments and cell lines\",\n      \"pmids\": [\"11504738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The interaction of CD81 with PGRL (EWI-2/IGSF8) requires the membrane-distal Ig3-Ig4 domains of PGRL, as determined by domain deletion constructs co-expressed with CD81.\",\n      \"method\": \"Co-expression of domain deletion constructs with CD81, immunoprecipitation\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping using epitope-tagged deletion constructs, single lab\",\n      \"pmids\": [\"11673522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Two separate regions of CD9 (40 and 47 amino acids) confer the ability to interact with EWI-2, as determined using chimeric CD9/CD82 molecules. EWI-2 was also shown to associate with CD81 on freshly isolated hepatocytes and to co-localize with CD81 in the liver.\",\n      \"method\": \"Chimeric CD9/CD82 protein expression, co-immunoprecipitation, immunofluorescence co-localization\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping with chimeric proteins, single lab, orthogonal localization data\",\n      \"pmids\": [\"12708969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EWI-2/PGRL (IGSF8) physically associates with the metastasis suppressor KAI1/CD82 in a highly stoichiometric, direct complex independent of cholesterol-enriched lipid rafts and distinct from CD81/CD9 complexes. Overexpression of EWI-2/PGRL in Du145 prostate cancer cells inhibits cell migration on fibronectin- and laminin-coated surfaces, and synergizes with KAI1/CD82 in migration suppression.\",\n      \"method\": \"Co-immunoprecipitation, chemical cross-linking, cell migration assays, overexpression\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP with cross-linking and functional migration assay, single lab\",\n      \"pmids\": [\"12750295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EWI-2 overexpression in A431 cells impairs cell reaggregation and motility on laminin-5 (an alpha3beta1 integrin ligand) but not on collagen I. Tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin. CD81 controls EWI-2 maturation and cell surface localization. A chimeric EWI-2 mutant failed to suppress migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation.\",\n      \"method\": \"Overexpression, immunodepletion, reexpression experiments, cell migration assays, mutagenesis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (immunodepletion, overexpression, mutant rescue, functional assays), mechanistic pathway placement established\",\n      \"pmids\": [\"14662754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EWI-2 is the most prominent alpha4beta1 integrin partner on MOLT-4 T leukemia cells. Wild-type EWI-2 overexpression markedly impairs cell spreading and ruffling on VCAM-1, while a cytoplasmic tail mutant EWI-2 neither impairs spreading nor associates with alpha4beta1 or CD81. EWI-2 overexpression reorganizes cell-surface CD81, increases CD81-CD81, CD81-alpha4beta1, and alpha4beta1-alpha4beta1 associations, and increases the apparent size of CD81-alpha4beta1 complexes.\",\n      \"method\": \"Anti-integrin antibody bead isolation, mass spectrometry, overexpression, cytoplasmic tail mutagenesis, shear flow adhesion assays, size exclusion chromatography\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mass spectrometric identification, multiple functional assays, mutagenesis confirming cytoplasmic tail requirement, single lab with orthogonal methods\",\n      \"pmids\": [\"15070678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EWI-2 (IGSF8) directly associates with ezrin-radixin-moesin (ERM) proteins through a basic charged amino acid stretch in its cytoplasmic domain. EWI-2 co-localizes with ERM proteins at microspikes, microvilli, and the cellular uropod in polarized leukocytes. Silencing EWI-2 augments cell migration, cellular polarity, and increases ERM phosphorylation. A dominant-negative moesin N-terminal domain delocalizes EWI-2 from the uropod. EWI-2 thereby links tetraspanin microdomains to the actin cytoskeleton.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, confocal microscopy, siRNA knockdown, dominant-negative overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding confirmed by GST pulldown, in vivo co-IP, dominant-negative functional validation, and siRNA phenotype with multiple orthogonal methods\",\n      \"pmids\": [\"16690612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EWI-2/CD316 (IGSF8) functions as a receptor for heat shock protein HSPA8 (hsp70 family member) on human dendritic cells. Soluble EWI-2 bound to cells expressing HSPA8 and to immobilized HSPA8 protein. HSPA8 ligation of EWI-2 enhanced CCL21/SLC-dependent migration of mature dendritic cells but attenuated their antigen-specific stimulatory capacity.\",\n      \"method\": \"Expression cloning, binding assays (soluble protein to cells and immobilized protein), functional migration assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — expression cloning identification of ligand plus direct binding assays and functional readouts, single lab\",\n      \"pmids\": [\"17785435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EWI-2 (IGSF8) overexpression in glioblastoma cell lines inhibits colony formation in soft agar and reduces cell motility and invasion. At the biochemical level, EWI-2 causes CD9 and CD81 to become more associated with each other and causes CD81 and other tetraspanins to become less associated with MMP-2 and MT1-MMP.\",\n      \"method\": \"Overexpression in T98G and U87-MG cells, soft agar assay, motility/invasion assays, co-immunoprecipitation, in vivo xenograft\",\n      \"journal\": \"Neoplasia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays plus co-IP evidence for molecular reorganization, single lab\",\n      \"pmids\": [\"19107234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EWI-2 (IGSF8) forms a direct, stable interaction with CD9 on mouse eggs (stable in 1% Triton X-100). Anti-IgSF8 antibody has moderate inhibitory effects on sperm-egg binding, whereas anti-CD9 antibody significantly inhibits sperm-egg fusion, indicating that IgSF8 and CD9 have discrete, non-identical functions in gamete interaction.\",\n      \"method\": \"Detergent co-immunoprecipitation, antibody perturbation in IVF assays\",\n      \"journal\": \"Reproduction, fertility, and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct co-IP under stringent detergent conditions and antibody functional assays with distinct phenotypic readouts\",\n      \"pmids\": [\"19210920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"On CD9-null oocytes, surface EWI-2 expression is reduced to <10% of wild-type levels, indicating that CD9 is required for normal surface localization of EWI-2. Depalmitoylated CD9 mutant does not affect EWI-2 surface levels, showing that CD9 palmitoylation is not required for maintaining EWI-2 at the surface.\",\n      \"method\": \"Flow cytometry on CD9 knockout oocytes, depalmitoylation mutant analysis\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with quantitative surface protein measurement, single lab\",\n      \"pmids\": [\"19107828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint is essential for interaction with CD81. Palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for interaction with both CD81 and CD9. EWI-2wint (a proteolytic cleavage product of EWI-2) must interact with CD81 to exert its inhibitory effect on HCV infection.\",\n      \"method\": \"Mutagenesis of transmembrane glycine zipper and cytoplasmic cysteine residues, co-immunoprecipitation, HCV infection functional assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis of specific motifs (glycine zipper, palmitoylation cysteines) with co-IP and functional HCV infection assays; multiple orthogonal mechanistic validations in single study\",\n      \"pmids\": [\"21343309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The EWI-2 cytoplasmic tail specifically interacts with negatively charged phosphatidylinositol phosphates (PIPs), particularly PtdIns5P, PtdIns4P, and PtdIns3P, but not other membrane lipids. Mutation of either of two basic residue clusters abolishes PIP binding. EWI-2 is constitutively palmitoylated at cytoplasmic cysteine residues. PIP binding regulates EWI-2 protein stability, while palmitoylation is required for tetraspanin-EWI-2 association and EWI-2-dependent inhibition of cell migration and lamellipodia formation.\",\n      \"method\": \"Lipid binding assays, site-directed mutagenesis of basic residue clusters and cysteines, palmitoylation assays, cell migration assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro lipid binding assays with mutagenesis, palmitoylation assays, and functional cellular readouts establishing differential roles of two modifications\",\n      \"pmids\": [\"21609323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EWI-2 co-localizes with CD3 and CD81 at the central supramolecular activation cluster (cSMAC) of the T cell immune synapse. Mass spectrometry of pull-downs with the C-terminal intracellular domain of EWI-2 identified α-actinin as a specific binding partner; this association is regulated by PIP2. Silencing EWI-2 or overexpression of a cytoplasmic-truncated EWI-2 mutant increases IL-2 secretion upon antigen stimulation. Silencing EWI-2 or α-actinin-4 increases HIV cell infectivity at virological synapses.\",\n      \"method\": \"Confocal microscopy, mass spectrometry pull-down, siRNA knockdown, overexpression of truncation mutant, IL-2 secretion assay, HIV infectivity assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mass spectrometric identification of binding partner, co-localization, mutagenesis, and multiple functional readouts (IL-2, HIV infection), single lab with orthogonal methods\",\n      \"pmids\": [\"22689882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"IGSF8 (EWI-2) interacts with tetraspanin CD9 in the olfactory bulb. Both IgSF8 and CD9 localize to puncta within axons and growth cones of olfactory sensory neurons, consistent with tetraspanin-enriched microdomain localization. IgSF8 expression in synaptic neuropil is transitory during glomerular formation and re-appears upon OSN regeneration, consistent with a role in synapse formation.\",\n      \"method\": \"Co-immunoprecipitation from olfactory bulb tissue, immunofluorescence, in vivo lesion/regeneration experiments\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP from tissue, in vivo regeneration experiment, single lab\",\n      \"pmids\": [\"22687584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Igsf8-deficient female mice show no fertilization defect in vitro or in vivo. Igsf8-deficient eggs retain normal CD9 levels and localization with normal microvilli formation, indicating IGSF8 is dispensable for fertility and not required for CD9 surface localization (contrary to a previous suggestion).\",\n      \"method\": \"Homologous recombination knockout, in vitro and in vivo fertility testing, immunofluorescence\",\n      \"journal\": \"Fertility and sterility\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with in vivo and in vitro functional tests; negative result rigorously established\",\n      \"pmids\": [\"22609062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EWI-2 (IGSF8) negatively regulates TGF-β signaling in melanoma. When EWI-2 is present, it sequesters CD9 and CD81 away from TGF-β receptors. When EWI-2 is knocked down, CD9 and CD81 become available to support TβR2-TβR1 association, elevating TGF-β signaling and downstream cytostasis, EMT-like changes, CD271-dependent invasion, and lung metastasis. All EWI-2 depletion phenotypes are reversed by blocking or depleting CD9 or CD81.\",\n      \"method\": \"RNAi knockdown, overexpression, co-immunoprecipitation, in vitro and in vivo functional assays, epistasis by CD9/CD81 blocking\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis established by rescue experiments (CD9/CD81 depletion reverses EWI-2 KD phenotype), multiple orthogonal functional readouts in vitro and in vivo\",\n      \"pmids\": [\"25656846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EWI-2 accumulates at the presynaptic terminal (producer cell side) of the HIV virological synapse and contributes to inhibition of Env-mediated cell-cell fusion. EWI-2 is downregulated upon HIV-1 infection, likely by Vpu. EWI-2 and CD81 surface levels are restored on syncytia where they continue to act as fusion inhibitors.\",\n      \"method\": \"Quantitative fluorescence microscopy, shRNA knockdown, cell-cell fusion assays\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — shRNA knockdown with cell-cell fusion functional assay and quantitative microscopy, single lab\",\n      \"pmids\": [\"31757023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"IgSF8 is a neuronal receptor enriched in the hippocampal mossy fiber (MF) pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Cell-surface interactome screening identified IgSF8 interaction partners at the MF synapse.\",\n      \"method\": \"MF synaptosome proteomic profiling, cell-surface interactome screening, conditional presynaptic knockout, electrophysiology, electron microscopy\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with multiple orthogonal readouts (structural, electrophysiological), interactome screening, single study with rigorous controls\",\n      \"pmids\": [\"33057002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EWI-2 (IGSF8) localizes not only on the plasma membrane but also on the nuclear envelope, where it regulates nuclear translocation of EGFR-MAPK-ERK signaling molecules and sorting of miR-3934-5p between cells and exosomes, thereby regulating prostate cancer cell metastasis.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence, exosome isolation, miRNA quantification, signaling pathway analysis\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, nuclear localization claim and miRNA sorting based on fractionation without functional mutagenesis; no mechanistic dissection of how nuclear EWI-2 regulates these processes\",\n      \"pmids\": [\"33605506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EWI2 (IGSF8) prevents EGFR from clustering and endocytosis, thereby restraining EGFR activation and ERK MAP kinase signaling. Upon EWI2 silencing or ablation, EGFR clustering, endocytosis, and activation increase, leading to enhanced cancer cell proliferation and migration. Inhibition of EGFR or ERK kinase abrogates the phenotypes caused by EWI2 removal.\",\n      \"method\": \"siRNA knockdown, CRISPR ablation, super-resolution microscopy (EGFR clustering), endocytosis assays, kinase inhibitor epistasis, in vivo xenograft\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR knockout plus pharmacological epistasis, direct measurement of EGFR clustering, single lab\",\n      \"pmids\": [\"35773608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EWI2 (IGSF8) promotes endolysosomal trafficking and lysosomal degradation of growth factor receptors and integrins by promoting nuclear retention of the master transcription factor TFEB, thereby driving lysosomogenesis and reducing surface levels of these receptors in lung cancer cells.\",\n      \"method\": \"Forced overexpression, lysosomal trafficking assays, TFEB nuclear localization experiments, receptor degradation assays, in vivo tumor models\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic linkage via TFEB nuclear retention with functional lysosomal degradation readouts, single lab\",\n      \"pmids\": [\"35339615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"IGSF8 expressed on tumors suppresses NK cell function by directly interacting with human KIR3DL2 and mouse Klra9 inhibitory receptors on NK cells. An antibody blocking IGSF8-NK receptor interaction enhances NK cell killing of malignant cells in vitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and T cell signaling in vivo. Anti-IGSF8 alone or combined with anti-PD1 inhibits tumor growth in syngeneic models.\",\n      \"method\": \"CRISPR screens, co-immunoprecipitation/binding assays, antibody blockade, NK cell killing assays, in vivo syngeneic tumor models\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen discovery followed by direct binding confirmation, antibody functional blockade, and in vivo validation in multiple tumor models\",\n      \"pmids\": [\"38657602\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IGSF8 (EWI-2) is a cell-surface immunoglobulin superfamily transmembrane protein that forms highly stoichiometric, direct complexes with tetraspanins CD9 and CD81 (requiring its Ig3-Ig4 domains and cytoplasmic palmitoylation), links tetraspanin-enriched microdomains to the actin cytoskeleton via direct binding to ERM proteins and α-actinin through its basic cytoplasmic tail, regulates integrin (α3β1, α4β1) and EGFR clustering and signaling, suppresses TGF-β signaling by sequestering CD9/CD81 away from TGF-β receptors, promotes lysosomal degradation of surface receptors via TFEB nuclear retention, inhibits HIV cell-cell fusion at virological synapses, functions as a presynaptic organizer of hippocampal CA3 mossy fiber microcircuit connectivity, and serves as an innate immune checkpoint by engaging inhibitory KIR3DL2/Klra9 receptors on NK cells to suppress anti-tumor cytotoxicity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IGSF8 (EWI-2) is a cell-surface immunoglobulin-superfamily transmembrane protein that organizes tetraspanin-enriched microdomains and couples them to the actin cytoskeleton and downstream receptor signaling [#0, #6]. It forms highly stoichiometric, direct, cross-linkable complexes specifically with the tetraspanins CD9 and CD81 (~70% of CD9/CD81 engaged), an interaction that requires its membrane-distal Ig3-Ig4 domains, a transmembrane glycine-zipper motif, and palmitoylation of juxtamembrane cytoplasmic cysteines [#0, #1, #11]. Through these tetraspanins IGSF8 is physically linked to integrins (α3β1, α4β1), and its overexpression reorganizes surface CD81-integrin complexes and suppresses cell spreading, migration, and invasion in a manner dependent on its cytoplasmic tail [#4, #5]. The basic cytoplasmic tail binds ERM proteins and α-actinin and engages phosphatidylinositol phosphates, tethering tetraspanin microdomains to the cytoskeleton at uropods and immune synapses; PIP binding stabilizes the protein while palmitoylation enables tetraspanin association and migration suppression [#6, #12, #13]. IGSF8 restrains receptor signaling on multiple axes: it sequesters CD9/CD81 away from TGF-β receptors to limit TGF-β-driven EMT and metastasis [#16], prevents EGFR clustering and endocytosis to dampen ERK signaling [#20], and promotes lysosomal degradation of growth-factor receptors and integrins via TFEB nuclear retention [#21]. It localizes to the central supramolecular activation cluster of the T-cell synapse and to HIV virological synapses, where it inhibits Env-mediated cell-cell fusion [#13, #17]. In neurons, IGSF8 acts as a presynaptic organizer of hippocampal mossy-fiber connectivity, controlling bouton filopodia and excitation/inhibition balance [#18]. As a tumor-expressed ligand it engages the inhibitory NK-cell receptors KIR3DL2 (human) and Klra9 (mouse) to suppress anti-tumor cytotoxicity, and antibody blockade of this interaction enhances NK killing and tumor control [#22].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established the founding molecular fact that IGSF8 is a dedicated, near-stoichiometric direct partner of specific tetraspanins, defining it as a core component of tetraspanin microdomains rather than a promiscuous associate.\",\n      \"evidence\": \"Co-purification under stringent Brij 96 detergent, mass spectrometry, and chemical cross-linking; domain-deletion mapping with CD81\",\n      \"pmids\": [\"11504738\", \"11673522\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the IGSF8-tetraspanin complex\", \"Stoichiometry of higher-order assemblies undefined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapped the binding determinants on the tetraspanin side and showed IGSF8 also partners the metastasis suppressor CD82, linking tetraspanin association to suppression of tumor cell migration.\",\n      \"evidence\": \"Chimeric CD9/CD82 constructs, reciprocal co-IP with cross-linking, hepatocyte co-localization, and migration assays in prostate cancer cells\",\n      \"pmids\": [\"12708969\", \"12750295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of migration suppression by IGSF8-CD82 not resolved\", \"Single-lab functional assays\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrated that IGSF8 reorganizes integrin-tetraspanin complexes and that this activity, and surface maturation, depend on CD81 and on the IGSF8 cytoplasmic tail.\",\n      \"evidence\": \"Overexpression, immunodepletion/reexpression, chimeric and cytoplasmic-tail mutants, migration and shear-flow adhesion assays for α3β1 and α4β1\",\n      \"pmids\": [\"14662754\", \"15070678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs tetraspanin-bridged contact with integrins not separated\", \"Signaling consequences downstream of integrin clustering undefined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified the cytoskeletal coupling mechanism, showing the basic cytoplasmic tail directly binds ERM proteins to tether tetraspanin microdomains to actin and restrain migration and polarity.\",\n      \"evidence\": \"GST pulldown, co-IP, confocal microscopy, siRNA knockdown, dominant-negative moesin in polarized leukocytes\",\n      \"pmids\": [\"16690612\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ERM binding is regulated by activation state unknown\", \"Relationship between ERM binding and integrin reorganization unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Proposed IGSF8 as a surface receptor for HSPA8 on dendritic cells with dual effects on migration and antigen presentation, broadening its role beyond tetraspanin scaffolding.\",\n      \"evidence\": \"Expression cloning, soluble-protein binding assays, dendritic cell migration and stimulation assays\",\n      \"pmids\": [\"17785435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site and physiological relevance of HSPA8 engagement unconfirmed\", \"Single-lab finding not extended\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined IGSF8-CD9 complexes in gametes and tested their reproductive role, with conflicting early conclusions about whether IGSF8 controls CD9 surface localization.\",\n      \"evidence\": \"Stringent detergent co-IP, antibody perturbation in IVF, flow cytometry on CD9-null oocytes\",\n      \"pmids\": [\"19210920\", \"19107828\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Apparent dependence of IGSF8 on CD9 later contradicted by knockout data\", \"Modest antibody effects on sperm-egg binding hard to interpret\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed IGSF8 overexpression suppresses glioblastoma colony formation and invasion by tightening CD9/CD81 association and reducing tetraspanin-MMP coupling, generalizing its tumor-suppressive microdomain remodeling.\",\n      \"evidence\": \"Overexpression in glioma lines, soft agar, motility/invasion assays, co-IP, xenografts\",\n      \"pmids\": [\"19107234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effect on MMP activity not measured\", \"Single-lab functional data\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the molecular grammar of tetraspanin binding and connected a proteolytic IGSF8 product to viral entry inhibition, defining the transmembrane and palmitoylation requirements.\",\n      \"evidence\": \"Mutagenesis of the transmembrane glycine zipper and juxtamembrane palmitoylation cysteines, co-IP, HCV infection assays; PIP-binding lipid assays and palmitoylation assays\",\n      \"pmids\": [\"21343309\", \"21609323\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How EWI-2wint cleavage is regulated unknown\", \"Distinct contributions of PIP binding vs palmitoylation to in vivo function not fully separated\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed IGSF8 at the immune and virological synapse via α-actinin/PIP2-regulated cytoskeletal coupling, where it dampens T-cell IL-2 output and restricts HIV infectivity.\",\n      \"evidence\": \"Confocal microscopy, mass-spectrometry pulldown identifying α-actinin, siRNA, truncation mutants, IL-2 and HIV infectivity assays; plus olfactory-bulb co-IP and regeneration data\",\n      \"pmids\": [\"22689882\", \"22687584\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect α-actinin contact in vivo unconfirmed\", \"Mechanism connecting synapse architecture to cytokine output unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Genetic knockout established that IGSF8 is dispensable for fertility and not required for CD9 surface localization, correcting an earlier interpretation.\",\n      \"evidence\": \"Homologous-recombination knockout with in vitro and in vivo fertility testing and immunofluorescence\",\n      \"pmids\": [\"22609062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address redundancy with other EWI proteins\", \"Reproductive role, if any, remains negative\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established a defined signaling mechanism: IGSF8 suppresses TGF-β signaling and metastasis by sequestering CD9/CD81 from TGF-β receptors, proven by CD9/CD81-dependent rescue.\",\n      \"evidence\": \"RNAi, overexpression, co-IP, epistasis via CD9/CD81 blockade, in vitro and in vivo melanoma metastasis assays\",\n      \"pmids\": [\"25656846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative basis of tetraspanin partitioning between receptor pools undefined\", \"Generality across other TGF-β receptor contexts untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended the fusion-inhibitory role to the HIV producer cell, showing IGSF8 is downregulated during infection (likely by Vpu) and inhibits Env-mediated cell-cell fusion.\",\n      \"evidence\": \"Quantitative fluorescence microscopy, shRNA, cell-cell fusion assays\",\n      \"pmids\": [\"31757023\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism of Vpu-mediated downregulation not demonstrated\", \"Single-lab functional data\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined a developmental neuronal function: presynaptic IGSF8 organizes mossy-fiber microcircuit architecture and excitation/inhibition balance.\",\n      \"evidence\": \"MF synaptosome proteomics, cell-surface interactome screening, conditional presynaptic knockout, electrophysiology, electron microscopy\",\n      \"pmids\": [\"33057002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trans-synaptic ligand mediating the organizing activity not pinpointed\", \"Relationship to tetraspanin partners in neurons unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Unified IGSF8's receptor-restraining activities, showing it limits EGFR clustering/endocytosis and drives TFEB-dependent lysosomal degradation of growth-factor receptors and integrins to suppress tumor cell proliferation.\",\n      \"evidence\": \"siRNA/CRISPR ablation, super-resolution imaging of EGFR clustering, endocytosis assays, kinase-inhibitor epistasis, TFEB nuclear-localization and receptor-degradation assays, xenografts\",\n      \"pmids\": [\"35773608\", \"35339615\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How IGSF8 signals to TFEB nuclear retention is unknown\", \"Direct mechanism preventing EGFR clustering not structurally defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified IGSF8 as a tumor-expressed innate immune checkpoint engaging inhibitory NK receptors KIR3DL2/Klra9, establishing a therapeutically actionable ligand-receptor axis.\",\n      \"evidence\": \"CRISPR screens, binding/co-IP, antibody blockade, NK killing assays, syngeneic tumor models with anti-PD1 combination\",\n      \"pmids\": [\"38657602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of IGSF8-KIR3DL2 recognition not defined\", \"Relationship between checkpoint function and tetraspanin scaffolding unexplored\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How IGSF8's diverse roles—tetraspanin scaffolding, cytoskeletal coupling, receptor signaling restraint, synaptic organization, and NK checkpoint ligand activity—are integrated by a single protein, and how these are regulated in different cell types, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of full-length IGSF8 in complex with any partner\", \"Whether the extracellular NK-receptor engagement and intracellular tetraspanin/cytoskeleton functions act independently is unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [16, 20, 21]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [6, 13]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [22]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [7, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4, 13]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [6, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [16, 20]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [13, 22]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [16, 22]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [20, 21]}\n    ],\n    \"complexes\": [\n      \"tetraspanin-enriched microdomain (CD9/CD81/EWI-2)\"\n    ],\n    \"partners\": [\n      \"CD81\",\n      \"CD9\",\n      \"CD82\",\n      \"ERM proteins\",\n      \"alpha-actinin\",\n      \"KIR3DL2\",\n      \"HSPA8\",\n      \"ITGA3\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}